Activin belongs to the transforming growth factor-β (TGF-β) superfamily and is a multifunctional growth-differentiation factor for differentiation induction of erythroblastic cells, secretion of follicle stimulating hormone (FSH) from the hypophysis, and promotion of luteinization caused by FSH, action to maintain the survival of nerve cells, and action to promote insulin production, for example.
Growth/differentiation factor-8 (GDF-8) has been discovered as a member of the TGF-β superfamily belonging to the activin subfamily and being specifically expressed during development and in the skeletal muscles of an adult (Nature, England, 1997, Vol. 387, No. 6628, pp. 83-90). GDF-8 is also referred to as myostatin and has a function to negatively control the muscle volume. In the case of a GDF-8-deficient mouse, the muscle volume increases to 2 to 3 times greater than that of a wild-type mouse (Nature, England, 1997, Vol. 387, No. 6628, pp. 83-90).
Follistatin binds strongly to activin (Science, U.S.A., 1990, Vol. 247, No. 4944, pp. 836-838) and to GDF-8 (Proceedings of the National Academy of Sciences of the United States of America, U.S.A., 2001, Vol. 98, No. 16, pp. 9306-9311) (dissociation constant to activin: Kd=540-680 pmol/L), and neutralize their activities. Furthermore, data has been accumulated that suggests direct and indirect in vivo involvement of follistatin in control of various physiological activities of activin and GDF-8. When follistatin is continuously administered to a mouse via an osmotic mini pump, the number of erythroblastic progenitor cells in the bone marrow or the spleen is significantly decreased (Proceedings of the National Academy of Sciences of the United States of America, U.S.A., 1992, Vol. 89, No. 5, pp. 1553-1556), and the skeletal muscle mass is significantly increased in the case of a transgenic mouse which expresses follistatin under control of a skeletal-muscle-specific promoter (Proceedings of the National Academy of Sciences of the United States of America, U.S.A., 2001, Vol. 98, No. 16, pp. 9306-9311).
Analysis of the amino acid sequence of follistatin has revealed that follistatin comprises 4 domains (from the N-terminal side): the N-terminal domain, follistatin domain I, follistatin domain II, and follistatin domain III (Proceedings of the National Academy of Sciences of the United States of America, U.S.A., 1988, Vol. 85, No. 12, pp. 4218-4222). The follistatin domains are thought to be involved in binding to activin or other growth factors.
In recent years, there have been reports suggesting that GDF-8 is involved in various diseases. Zimmers et al. have demonstrated that transplantation of cells stably expressing GDF-8 into the femoral region of a nude mouse results in induction of cachexic symptoms including decreased skeletal muscle and fat (Science, U.S.A., 2002, Vol. 296, No. 5572, pp. 1486-1488). An increased amount of anti-GDF-8-antibody-positive protein in the muscle of an HIV patient with body weight loss has also been reported (Proceedings of the National Academy of Sciences of the United States of America, U.S.A., 1998, Vol. 95, No. 25, pp. 14938-14943). It has also been reported that in the case of mice obtained through mating mdx mice, the model mice of Duchenne muscular dystrophy, with GDF-8-deficient mice, both muscle mass and muscle functions are improved as compared with those of mdx mice (Annals of Neurology, U.S.A., 2002, Vol. 52, No. 6, pp. 832-836). Moreover, since decreases in fat levels as well as increases in skeletal muscle are observed in the case of GDF-8-deficient mice and GDF-8 gene deficiency caused in diabetes model mice results in suppression of fat accumulation and abnormal glucose metabolism, involvement of GDF-8 in the onset of or progression of pathological conditions of obesity or Type II diabetes has also been suggested (The Journal of Clinical Investigation, U.S.A., 2002, Vol. 109, No. 5, pp. 595-601).
As described above, it is thought that through inhibition of GDF-8 activity, these diseases, to the onset or the progress of the pathological conditions of which GDF-8 contributes, can be treated. Moreover, for alleviating side effects, it is preferable to selectively inhibit GDF-8 activity without inhibiting activin activity. As substances that inhibit GDF-8 activity, follistatin (Description of U.S. patent application publication No. 2002/0157126 and JP Patent Publication (Kohyo) No. 2002-506044 A), an anti-GDF-8 antibody (Description of U.S. Pat. No. 6,096,506), a GDF-8 propeptide (International Patent Publication No. 02/09641 pamphlet), a dominant negative GDF-8 variant (International Patent Publication No. 01/53350 pamphlet, and International Patent Publication No. 00/43781 pamphlet), a GDF-8 antigen (Description of U.S. Pat. No. 6,369,201, International Patent Publication No. 01/05820 pamphlet, and International Patent Publication No. 99/42573 pamphlet), a GDF-8 receptor and an anti-GDF-8 receptor antibody (International Patent Publication No. 02/10214 pamphlet), and a Smad2 or Smad3 phosphorylation inhibitor (International Patent Publication No. 02/055077 pamphlet), for example, are known.